The use of stains is very important in the investigation of biological tissues and is of particular importance in hematology. By the use of appropriate stains, the various components of blood, such as platelets, cells and structures thereof may be selectively visualized, facilitating microscopic inspection of the blood sample and the identification of deviant morphologies and/or disease causing organisms. There are a variety of stains utilized in hematology. These stains are typically organic dyes having differential affinities for various cells and cell structures. One of the most commonly employed stains is what is referred to as Wright's stain. This stain, also called RG stain in Europe is a mixture of dyes prepared by the steps of oxidizing methylene blue and precipitating the mixture with tetrabromofluorescein. The preparation of this mixture of dyes was first described by J. H. Wright in the Journal of Medical Research, Volume 7, p. 138, 1902. Wright's stain is typically provided in the form of a prepared powder referred to as "Wright's stain powder". This is a staple item of commerce available from a variety of suppliers including the Aldrich Chemical Corporation of Milwaukee, Wis.
Commercially available Wright's stain is a complex mixture of many different dyes some of which are active and some of which are inactive, although it is generally agreed that the most important components are the azure A and azure B dyes. It has generally been found that the best Wright's stain usually has a first absorption maximum between 650-656 nm and a separate Eosin absorption peak around 517 nm although these peaks may differ significantly from batch to batch of dyes without significantly impairing stain function. It should be clear from the foregoing that the exact composition of a batch of Wright's stain powder is variable and is usually not precisely determined. For this reason, the term "Wright's stain powder" as utilized herein is generally meant to refer to methylene blue derived stain mixtures as are well-known and available to those of skill in the art and as such is a generic term and not restricted to any one specific dye mixture.
Although Wright's stain has been known for some time, its use is not without problems. It has previously been found that Wright's stain suffers from problems of stability, both of the prepared solution and the sample stained thereby. U.S. Pat. No. 3,670,072, the disclosure of which is incorporated herein by reference, recognizes these problems of stability and provides for an improved stain system incorporating Wright's stain powder together with buffers and anti-oxidant fixatives. The invention of the '072 patent provides for a fast acting and permanent staining system.
Despite these advances, there are still problems associated with Wright's stain materials. For example, it has been found that in many instances the granulation of stained white blood cells is indistinct. This effect correlates with particular batches of Wright's stain powder and also appears to be related to the pH of the blood samples. Incorporation of the afore-mentioned buffers does not fully address problems of indistinct granulation and further investigation suggests that the pH of the dried blood samples may affect the differentiation of the granulation. Another problem with stain materials of this type is that the color imparted to the blood cells is frequently too blue thereby preventing resolution of fine detail and differentiation of cell structures. It is believed that this problem also relates inter alia to inconsistencies in the commercially available Wright's stain powder. These problems are particularly important now that computerized interpretation of blood smears is coming into use. The use of such automated methods exacerbates the need for quicker and more uniform staining of blood smears. Toward that end, there is a need for improved wetting agents.
Yet another problem which occurs in connection with the use of various stain materials is the presence of artifacts therein. These artifacts comprise particulate contaminants in the stain solution, which may be undissolved dye particles, trace contaminants and the like. These artifacts can interfere with interpretation of test results and are particularly troublesome in connection with automated evaluation systems.
It will thus be appreciated that there is a need for a Wright's stain system which enhances the structure of the granulation of white blood cells and improves the overall differentiation of cell structure by eliminating the effects of any "blue shift". It is further desired that these Wright's stain systems include a low number of artifacts and uniformly and rapidly wet and stain blood samples. The present invention provides an improved Wright's stain system which: greatly enhances the details of cell granulation, provides a color balance which maximizes differentiation of cell structures, is low in artifacts and rapidly and consistently stains a variety of blood samples. These and other advantages of the present invention will be readily apparent from the discussion and examples which follow.